Field of the Invention
This invention relates to the preparation of water-insoluble composites of the coenzyme nicotinamide-adenine-dinucleotide (NAD) and various carrier materials. More specifically, the invention relates to methods of insolubilizing NAD in such a manner that the coenzyme can be used repeatedly in chemical reaction.
A coenzyme, sometimes referred to as a cofactor, is a thermostable nonprotein compound of relatively low molecular weight which is essential for the catalytic action of many enzymes. Enzymes are biological catalysts capable of initiating, promoting, and governing chemical reactions. All known enzymes are proteins and it is well known that they are essential for numerous biochemical reactions. Typically, an enzyme reacts with one or more substrates to produce products without being used up in the process or becoming a part of the products formed. In many enzymatic reactions, the presence of an active coenzyme is required. Examples of well known coenzymes are nicotinamide-adenine-dinucleotide (NAD), flavin-adenine-dinucleotide (FAD), and adenosine-triphosphate (ATP).
Nicotinamide-adenine-dinucleotide acts as a proton acceptor in its oxidized form (NAD) and as a proton donor in its reduced form (NADH). NAD and NADH have the following chemical structures: ##SPC1##
NAD and other coenzymes are of great practical interest because of the essential roles they play in biochemical processes. For example, the interior of living cells is filled with many particulate structures, each having various functions to perform. One group of structures, the mitochondria, have as a primary function the oxidation of succinic acid and NADH and the coupling of those oxidations to the esterification of adenosine-diphosphate (ADT) to the triphosphate (ATP). Also, four of the five oxidation steps involved in the combustion of pyruvate to carbon dioxide and water consist of dehydrogenation in which hydrogen is transferred from the substrate to the oxidized coenzyme, NAD. The NADH so formed is reoxidized by a flavoprotein enzyme system in yet another biochemical reaction. Thus, it can be readily appreciated that NAD, with other coenzymes, are essential compounds in life processes. Consequently, considerable attention has been directed toward further understanding the roles coenzymes play in biological systems.
Because many enzyme systems require the presence of NAD or NADH, there are many present and potential applications in research, analytical procedures, and industry in which the coenzyme is and can be utilized. For example, it is known that many enzymes utilizing NAD exhibit reasonable binding constants for the nucleotide. Consequently, enzyme purification techniques would be possible if there were methods available for retaining the coenzyme in a column through which solutions known to contain certain enzymes could flow. Further, since the coenzyme is spectrophotometrically detectable, it can be used for the quantitative and qualitative analysis of various products in enzymatic reactions which require NAD or NADH since the coenzyme is commonly either oxidized or reduced in direct proportion to the amount of product produced or the amount of substrate transformed. Also, metabolic pathways may be constructed if there were methods for modifying the coenzyme in such a way that it could be made either water-insoluble or retainable within membranes without loss of its redox properties. The above applications are but a few of the many present and potential applications in which NAD is and will be essential.
There are, however, certain disadvantages accompanying the use of NAD which greatly limit its present and potential value as a research and industrial tool. For example, in many enzymatic reactions, the cost of the coenzyme exceeds the cost of the enzyme. Although some enzymes can be recovered for repeated use, the coenzyme is difficult to recover. Consequently, the more costly NAD can generally be utilized only once. Also, even though NAD is relatively stable in pure form, it is frequently rendered unstable or inactive by the presence of the other compounds with which it may be stored or used. Thus, further industrial uses of enzymatic reactions requiring NAD are limited by the high cost, unreusability, and the frequent instability of NAD.
Prior Art
Recently, there have been devised methods for rendering otherwise soluble enzymes water-insoluble by attachment to water-insoluble carriers in such a manner that the enzymes retain their catalytic power. For example, U.S. Pat. No. 3,519,538 teaches methods for chemically coupling enzymes to inorganic carriers in such a way that the enzymes remain catalytically active so they can be used repeatedly. Also, U.S. Pat. No. 3,556,945 teaches methods for absorbing enzymes to inorganic carriers such as glass particles. There has also been recently disclosed a method for stabilizing reduced NAD (NADH) by combining it with SH-group containing compounds. See Canadian Patent No. 862,061. In that disclosure, however, the reduced NAD is not rendered water-insoluble for repeated use but, rather, it is stabilized to the extent that it does not lose its activity when in contact with other compounds. Thus, even though the costly reduced form of NAD can be utilized more efficiently according to the above teaching, and even though it is known that NAD can be made more efficiently (see, for example, Canadian Pat. No. 868,877), the coenzyme can generally be used only once without further elaborate recovery procedures.
To date, there have been no known methods for overcoming the disadvantages associated with the coenzyme. Thus, there has been a continuing need for methods of modifying NAD in such a manner that the coenzyme is made water-soluble for repeated, economical use and stabilized in a biologically active form. The present invention, quite surprisingly, serves that need.